Staphylococcus aureus is an important human pathogen that has accumulated resistance to multiple antibiotics, including in methicillin-resistant and vancomycin- intermediate strains. Various antimicrobial substances have important roles to play in chemical control of the organism including disinfectant, antiseptics, innate and exogenous antimicrobial peptides, and antibiotics. Many of the antimicrobials target or interact with the cytoplasmic membrane of the organism. Surprisingly, evidence has been presented that decreased susceptibility to two antimicrobial peptides, and organic solvents, is associated with increased membrane fluidity. This is a poorly studied but potentially of wide-ranging importance mechanism of antimicrobial resistance. In this AREA proposal we propose to examine the hypothesis that increased/decreased membrane fluidity plays an important role in decreased/increased susceptibility to membrane antimicrobials. In specific aim 1 a branched-chain fatty acid-deficient mutant with significantly decreased membrane fluidity will be used to study the impact of membrane fluidity on antimicrobial susceptibility. Poorly studied membrane lipid components in terms of their impact on membrane fluidity are carotenoids, the production of which is a hallmark of S. aureus. In specific aim 2 the impact of carotenoids on membrane fluidity and antimicrobial susceptibility will be studied using carotenoid-deficient mutants. In specific aim 3 we will attempt to answer the question as to whether strains resistant to membrane antimicrobials show increased membrane fluidity. This will be studied in pine oil cleaner- and daptomycin-resistant strains. Completion of the proposed research should further the aims of the AREA program, and elucidate in detail the role of membrane fluidity in the susceptibility of S. aureus to antimicrobial agents. Agents decreasing membrane fluidity may enhance the activity of membrane antimicrobials against S. aureus. PUBLIC HEALTH RELEVANCE: Antimicrobial-resistant strains of Staphylococcus aureus are a major public health threat. We are investigating a novel mechanism of resistance to antimicrobials targeting the membrane. We hope that the research will result in improved control of S. aureus.